To provide a plant with a novel character by transferring a gene into the plant is called transformation. When the gene transferred is expressed in plant cells, the character provided manifests itself. Once the gene has been integrated in an intracellular chromosome, the character provided will be maintained stably. Such character to be newly provided by gene transfer includes, for example, resistance to diseases and agricultural chemicals and changes in metabolism. Genes for use in such transformation can freely be constructed using the current gene recombination technology. Several methods have been developed for transferring the genes constructed in such a manner into plants. For efficiently integrating a gene into a plant cell nuclear chromosome, there is available the Agrobacterium infection method which utilizes, as a vehicle (vector) for the gene, Agtrobacterium, which is a plant-infective bacterium.
The expression of a gene involves a step, called transcription, in which mRNA is transcribed upon a template, namely DNA which is the very gene containing a genetic information, and a step, called translation, in which a protein is synthesized based on the genetic information from the transcript mRNA. It is known that a gene comprises regions involved in transcriptional regulation or control in addition to the region encoding the protein information. The most basic transcriptional regulatory region is a 5′ upstream region relative to the coding region and is called a promoter. The promoter differs in structure between eukaryotes, such as plants, and prokaryotes, such as bacteria. Plant promoters have a nucleotide sequence called TATA box, which is essential for initiating gene transcription, and other various regulatory sequences. For initiating transcription, RNA polymerase, which is an enzyme catalyzing the transcription in plant cells, binds to the TATA box. Various intracellular proteins called transcription factors specifically bind to the various regulatory sequences serving as targets for those factors. These transcription factors promote or inhibit the transcriptional activity of RNA polymerase and thereby control the gene expression. Thus, gene expression is under the control of such regulatory sequences. These regulatory sequences and transcription factors are also involved in induction of gene expression via the step of transcription.
To control the induction of expression of a gene transferred into a plant for transformation with respect to time and site makes it possible, with great advantage, to produce, in plants, such metabolites as otherwise will be disadvantageous to plant growth. For such purposes, the utilization of a gene expression inducing system of other organisms has often been attempted. This is because the use of a gene expression inducing system intrinsic in a plant as it is may possibly exert an unexpected influence on the metabolic system of the plant. However, it is not self-evident whether the gene expression inducing system of other organisms can be successfully given to the plant.
The regulatory system comprising an inducer, repressor and operator as found in the bacterial operon regulatory system is one of the principal gene expression inducing systems. The inducer is a low-molecular-weight compound inducing gene expression. The repressor is a receptor protein for the inducer. The operator is a regulatory sequence serving as a target for the repressor. The inducer-repressor binding and repressor-operator binding are very specific and show high levels of affinity, whereas the inducer-bound repressor cannot bind to the operator. A gene containing the operator in its promoter, namely a gene under the control of the operator, is inhibited (OFF) from being expressed when the inducer concentration is low because the repressor is bound to the operator but, as the inducer concentration increases, the repressor is released and gene expression is induced (ON).
Attempts have been reported to utilize the bacterial inducer/repressor/operator system as means for inducing gene expression in plants. For providing a plant with the characters of a repressor and operator, two genes, namely a repressor gene and a gene under the control of an operator, are transferred into the plant. For attaining expression of both genes in plant cells, it is desirable that the promoter therefor be a plant promoter. The operator is located in and near the plant promoter. By choosing the promoter, it is possible to functionally combine various characteristics of the promoter, such as gene expression intensity and tissue specificity, with the gene expression inductivity. By administering an inducer to the plant transformed in this manner, the expression of the gene placed under the control of the operator is induced at the site of administration of the inducer. As examples of the success in providing plants with such inducer/repressor/operator regulatory systems, there are reports on the systems in which tetracycline and IPTG are used as inducers [Japanese Kokai Publication Hei-06-339384 and Gatz et al., Trends in Plant Science (1998), 3, 352–358]. However, the inducer substances used in the examples so far reported have problems from feasibility points of view, for example in the aspects of environmental safety and/or cost of use.